The present invention is related to substituted imidazo[1,5-a]pyrimido[5,4-d][1]benzazepine derivatives of formula 
Compounds of formula 1 are ligands for GABA A receptors containing the xcex15 subunit and are therefore useful in therapy where cognition enhancement is desirable.
Receptors for the major inhibitory neurotransmitter, gamma-aminobutyric acid (GABA), are divided into two main classes: (1) GABA A receptors, which are members of the ligand-gated ion. channel superfamily and (2) GABA B receptors, which are members of the G-protein linked receptor family. The GABA A receptor complex which is a membrane-bound heteropentameric protein polymer is composed principally of xcex1, xcex2 and xcex3 subunits.
Presently a total number of 21 subunits of the GABA A receptor have been cloned and sequenced. Three types of subunits (xcex1, xcex2 and xcex3) are required for the construction of recombinant GABA A receptors which most closely mimic the biochemical, electrophysiological and pharmacological functions of native GABA A receptors obtained from mammalian brain cells. There is strong evidence that the benzodiazepine binding. site lies between the cc and y subunits. Among the recombinant GABA A receptors, xcex11xcex22xcex32 mimics many effects of the classical type-I BzR subtypes, whereas xcex12xcex22xcex32, xcex13xcex22xcex32 and xcex15xcex22xcex32 ion channels are termed type-II BzR.
It has been shown by McNamara and Skelton in Psychobiology, 21:101-108 that the benzodiazepine receptor inverse agonist xcex2-CCM enhance spatial learning in the Morris watermaze. However, xcex2-CCM and other conventional benzodiazepine receptor inverse agonists are proconvulsant or convulsant which prevents their use as cognition enhancing agents in humans. In addition, these compounds are non-selective within the GABA A receptor subunits, whereas a GABA A xcex15 receptor partial or full inverse agonist which is relatively free of activity at GABA A xcex11 and/or xcex12 and/or xcex13 receptor binding sites can be used to provide a medicament which is useful for enhancing cognition with reduced or without proconvulsant activity. It is also possible to use GABA A xcex15 inverse agonists which are not free of activity at GABA A xcex11 and/or xcex12 and/or xcex13 receptor binding sites but which are functionally selective for xcex15 containing subunits. However, inverse agonists which are selective for GABA A xcex15 subunits and are relatively free of activity at GABA A xcex11, xcex12 and xcex13 receptor binding sites are preferred.
The present invention is a compound of formula 
wherein
R1 is selected from the group consisting of halogen and lower alkyl;
R2 is selected from the group consisting of hydrogen, lower alkyl, cycloalkyl, xe2x80x94(CH2)m-phenyl, xe2x80x94(CH2)m-phenyl substituted by lower alkoxy and xe2x80x94(CH2)m-indolyl;
R3 is selected from the group consisting of xe2x80x94C(O)O-lower alkyl, xe2x80x94C(O)OH, a five-membered heteroaromatic group and a five-membered heteroaromatic group substituted by lower alkyl or cycloalkyl;
n is 0, 1 or 2; and
m is 0, 1 or 2;
or a pharmaceutically acceptable acid addition salt thereof
It has now been found that a compound of formula 1 shows high affinity and selectivity for GABA A xcex15 receptor binding sites and might be useful as a cognitive enhancer or for the treatment of cognitive disorders like Alzheimer""s disease.
An object of the present invention is compounds of formula I and pharmaceutically acceptable salts thereof. A further object of the invention is the preparation of the compounds of formula I and pharmaceutically acceptable salts thereof. Pharmaceutical compositions containing a therapeutically effective amount of the compound of formula I and salts thereof and their manufacture are objects of the present invention. Another object of the invention is a method of treatment for modulating GABA A xcex15 receptor binding sites as a cognitive enhancer or treatment of cognitive disorders like Alzheimer""s disease comprising the administration of a therapeutically effective amount of the compounds of formula I or a pharmaceutically acceptable salt. The most preferred indication in accordance with the method of treatment of the present invention is the treatment of cognitive disorders, like Alzheimer""s disease.
The following definitions of the general terms used in the present description apply irrespective of whether the terms in question appear alone or in combination.
As used herein, the term xe2x80x9clower alkylxe2x80x9d denotes a straight-or branched-chain alkyl group containing from 1-7 carbon atoms, for example, methyl, ethyl, propyl, isopropyl, n-butyl, i-butyl, t-butyl and the like. Preferred lower alkyl groups are groups with 1-4 carbon atoms.
The term xe2x80x9clower alkoxyxe2x80x9d denotes a croup wherein the alkyl residues are as defined above, and which is attached via an oxygen atom.
The term xe2x80x9chalogenxe2x80x9d denotes chlorine, iodine, fluorine and bromine.
The term xe2x80x9ccycloalkylxe2x80x9d denotes a cyclic alkyl ring, having from 3 to 7 carbon ring atoms, for example, cyclopropyl, cyclopentyl or cyclohexyl.
The term xe2x80x9cfive-membered heteroaromatic groupxe2x80x9d denotes, for example 1,2,4-oxadiazoles, furyl, pyrrolyl, thienyl, imidazolyl, pyrazolyl, isothiazolyl, isoxazolyl and the like. Preferred are 1,2,4-oxadiazolyl and isoxazolyl groups.
The term xe2x80x9cpharmaceutically acceptable acid addition saltsxe2x80x9d embraces salts with pharmaceutically acceptable inorganic and organic acids, such as hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, citric acid, formic acid, fumaric acid, maleic acid, acetic acid, succinic acid, tartaric acid, methane-sulfonic acid, p-toluenesulfonic acid and the like.
Exemplary preferred are compounds, which have a binding activity (Ki) of lower 15 nM and are selective for GABA A xcex15 subunits and are relatively free of activity at GABA A xcex11, xcex12 and xcex13 receptor binding sites.
Preferred compounds of formula I are those, in which R3 is the group xe2x80x94C(O)O-lower alkyl. Exemplary preferred are compounds of this group, wherein R1 is hydrogen and R2 is as described above, for example compounds selected from the group consisting of:
9H-imidazo[1,5-a]pyrimido[5,4-d][1]benzazepine-10-carboxylic acid ethyl ester,
6-propyl-9H-imidazo[1,5-a]pyrimido[5,4-d][1]benzazepine-10-carboxylic acid ethyl ester,
6-(1-methylethyl)-9H-imidazo[1,5-a]pyrimido[5,4-d][1]benzazepine-10-carboxylic acid ethyl ester,
6-cyclopropyl-9H-imidazo[1,5-a]pyrimido[5,4-d][1]benzazepine-10-carboxylic acid ethyl ester,
6-[(4-methoxyphenyl) methyl]-9H-imidazo[1,5-a]pyrimido[5,4-d][1]benzazepine-10-carboxylic acid ethyl ester and
6-methyl-9H-imidazo[1,5-a]pyrimido[5,4-d][1]benzazepine-10-carboxylic acid ethyl ester.
Further preferred compounds of formula I are those, in which R3 is the group xe2x80x94C(O)O-lower alkyl, R2 is as described above and R1 is halogen, for example compounds selected from the group consisting of:
3-fluoro-6-methyl-9H-imidazo[1,5-a]pyrimido[5,4-d][1]benzazepine-10-carboxylic acid ethyl ester,
3-fluoro-6-propyl-9H-imidazo [1,5-a]pyrimido[5,4-d][1]benzazepine-10-carboxylic acid ethyl ester,
3-fluoro-6-(1-methylethyl)-9H-imidazo[1,5-a]pyrimido[5,4-d][1]benzazepine-10-carboxylic acid ethyl ester,
6-cyclopropyl-3-fluoro-9H-imidazo[1,5-a]pyrimido[5,4-d][1]benzazepine-10-carboxylic acid ethyl ester and
3-bromo-6-methyl-9H-imidazo[1,5-a]pyrimido[5,4-d][1]benzazepine-10-carboxylic acid ethyl ester.
Further preferred compounds of formula I are those, in which R3 is the group 1,2,4-oxadiazolyl or isoxazolyl, R2 is lower alkyl, n is 0 or 1 and R1 is halogen, for example compounds selected from the group consisting of:
10-(3-cyclopropyl-1,2,4-oxadiazol-5-yl)-6-methyl-9H-imidazo[1,5-a]pyrimido[5,4-d][1]benzazepine and
2-bromo-11-methyl-7-(5-methyl-isoxazol-3-yl)-8H-4b,6,10,12-tetraaza-dibenzo [e,g]azulene.
The present compounds of formula I and their pharmaceutically acceptable salts may be prepared by methods known in the art, for example, by processes described below, reacting a compound of formula 
with phosphoroxychloride forming a compound of formula 
wherein the substituents R1 and R2 and n have the significances given above, and reacting this compound with 
forming a compound of formula 
and cyclizing this compound with
MeCO2H 
forming a compound of formula 
wherein R1-R3 and n have the significances given above, or reacting a compound of formula 
wherein the substituents R1 and R2 and n have the significances given in claim 1, with 
forming a compound of formula I, or modifying one or more substituents R1-R3 within the definitions given above, and if desired, converting the compounds obtained into a pharmaceutically acceptable acid addition salt.
The salt formation is effected at room temperature in accordance with known methods that are familiar to any person skilled in the art. Not only salts with pharmaceutically acceptable inorganic acids, but also salts with pharmaceutically acceptable organic acids are possible. Hydrochlorides, hydrobromides, sulfates, nitrates, citrates, acetates, maleates, succinates, methanesulfonates, p-toluenesulfonates and the like are examples of such salts.
The following schemes 1, 1a, 2, 3, 4, 5 and 6 describe in more detail the process for preparation of compounds of formula I and/or their intermediates. The starting materials of formulas IV, VI, XVI, XX and XXVII are known compounds or maybe prepared according to methods known in the art. 
The substituents given in scheme 1 are described above. 
The substituents given in scheme 1a are described above.
Phosphoroxychloride may be replaced by the following equivalent compounds: 
or methylsulfides in accordance with the following references:
J. Heterocycl. Chem., 1978, 15, 577-583
J. Org. Chem., 1976, 41, 2724-2727
J. Org. Chem., 1976, 41, 2720-2724 or
Synthesis, 1987, 162.
In accordance with schemes 1 and 1a a compound of formula I maybe prepared as follows: Starting from an appropriately substituted anthranilic acid (VI) the ester (VII) is prepared under standard conditions. Treatment of this product with an appropriate base and ethyl succinyl chloride to give the product (VIII) which is then reacted in an intramolecular Dieckmann cyclization to give the beta-keto ester products (IX). These are then de-ethoxy carboxylated under acidic or basic conditions to give the appropriately substituted benzazepinediones (X). Treatment of these products with dimethylformamide dimethoxy acetal provides the enaminone products (XI) which are then successively transformed to the 5,7-dihydro-6H-pyrimido[5,4-d][1]benzazepin-6-ones (II) by treatment with the appropriately substituted amidines (sometimes as salts) in the presence of sodium methoxide. The obtained compounds are then dissolved in phosphorus oxychloride and the solution heated and then evaporated. Then a solution of this product is added to a cold solution of either 1) ethyl isocyanoacetate and potassium tert-butoxide or 2) lithium diisopropylamide and (E)-(dimethylamino-methyleneamino)-acetic acid ester; and in a further step cyclized with addition of acetic acid followed by heating, The final products of formula I are purified in the conventional manner. 
This process has also been described in J. Heterocyclic Chem., 1965, 2, 459. 
The substituents given in scheme 3 are described above.
A mixture of xcex1-tetralone of formula XVI, hydroxylamine, sodium acetate and water/ethanol is treated under reflux for about 20 min and then cooled to 0xc2x0 C. The obtained product is added to a solution of polyphosphoric acid at about 120xc2x0 C. and heated. The lactam is then dissolved in BuOH and water, and then potassium permanganate is added followed by magnesium nitrate hexahydrate. This reaction is carried out at room temperature for about 48 h. A compound of formula Ia is then obtained followed by steps 5, 6 and 7 of scheme 1. 
The substituents given in scheme 4 are described above.
The preparation of these intermediates is described in more detail in the working examples. 
In accordance with scheme 5, a compound of formula IVb has been prepared, which is used for the preparation of compounds of formula I, wherein R3 is an isoxazole group. This reaction is described in scheme 6.
In accordance with scheme 5, the following reaction steps are described in more detail:
Step 1: 5-Methyl-isoxazole-3-carboxylic Acid Ethyl Ester
To a solution of ethyl-2,4-dioxovalerate in ethanol is added hydroxylamine hydrochloride and sodium hydrogen carbonate. The reaction mixture is then heated under reflux for 1 hour. After cooling, the mixture is evaporated to leave a clear liquid that was distilled to leave the title compound.
Step 2: (5-Methyl-isoxazol-3-yl)-methanol
To a solution of 5-methyl-isoxazole-3-carboxylic acid ethyl ester in ethanol under argon at 0xc2x0 C. is added portion wise NaBH4 over 30 minutes. The reaction is allowed to warm up to room temperature (rt). After 3 h the reaction mixture is diluted with HCl and then after cooling to room temperature the mixture is washed with ether, the combined extracts are dried and evaporated.
Step 3: 3-Bromomethyl-5-methyl-isoxazole
To a solution of PBr3 and pyridine in toluene is added at xe2x88x9210xc2x0 C. a solution of hydroxymethyl-3-methyl-5-isoxazote in pyridine. The reaction mixture is then stirred at xe2x88x9210xc2x0 C. for 1 h and stirred for about 14 h at rt. Then, the reaction mixture is diluted with water and extracted With ether. The combined extracts are then dried and evaporated. The residue is purified by chromatography.
Step 4: 3-Azidomethyl-5-methyl-isoxazole
To a solution of the 3-bromomethyl-5-methyl-isoxazole in acetone is added NaN3 at rt. The reaction mixture is then stirred for about 48 h. Then, the reaction mixture is poured into water and extracted with EtOAc, dried and evaporated.
Step 5: (5-Methyl-isoxazol-3-yl)-methylamine
To a solution of the 3-azidomethyl-5-methyl-isoxazole in isopropanol at rt with vigorous stirring is added triethylamine, 1,3 propanedithiol and sodium borohydride. The mixture is then stirred at rt. After about 19 hours 0.5 eq more of NaBH4 is added and stirred at rt for 7 hours more. Then the solvent is evaporated under vacuum and the residue is then dissolved in 10% aqueous citric acid and washed. The aqueous layer is basified with aqueous NaOH until pH 12, saturated with NaCl, and extracted with DCM. The combined DCM extracts are dried and concentrated.
Step 6: N,N-Dimethyl-Nxe2x80x2-(5-methyl-isoxazol-3yl-methyl)-formamidine
A solution of the (5-methyl-isoxazol-3-yl)-methylamine in N,N-dimethylformamide dimethylacetal is heated under reflux for 3 h. After cooling to room temperature, the solvent is evaporated to leave the compound of formula IVa.
The compound of formula IVa may then be added to a compound of formula III according to schemes 1a and 6. 
R1, R2 and n are described above.
As mentioned earlier, the compounds of formula I and their pharmaceutically acceptable salts possess valuable pharmacological properties. It has been found that the compounds of the present invention are ligands for GABA A receptors containing the xcex15 subunit and are therefore useful in the therapy where cognition enhancement is required.
The compounds of formula I were investigated in accordance with the test given hereinafter.
The affinity of compounds at GABA A receptor subtypes was measured by competition for [3H]flumazenil ([3H]Ro 15-1788) (85 Ci/mmol; Amersham) binding to SF9 cells expressing rat receptors of composition xcex11xcex23xcex32, xcex12xcex23xcex32, xcex13xcex23xcex32 and xcex15xcex23xcex32.
Cell pellets were suspended in Krebs-tris buffer (4.8 mM KCl, 1.2 mM CaCl2, 1.2 mM MgCl2, 120 mM NaCl, 15 mM Tris; pH 7.5; binding assay buffer), homogenized by polytron for ca. 15 sec on ice and centrifuged in UZ for 30 min at 4xc2x0 C. (100000 g; rotor: TFT 4594=300,000 rpm). The cell pellets were re-suspended in Krebs-tris buffer and homogenized by polytron for ca. 15 sec on ice. Aliquots of 1 ml were prepared, protein was measured (Bradford method) and the resulting membrane aliquots were stored at xe2x88x9270xc2x0 C.
Radioligand binding assays were carried out in a volume of 200 xcexcL (96-well plates) which contained 100 xcexcL of cells, [3H]Ro 15-1788 at a concentration of 1 nM for (xcex11, xcex12, xcex13 subunits and 0.5 nM for xcex15 subunits and the test compound in the range of 10xe2x88x9210xe2x88x923xc3x9710xe2x88x926 M. Nonspecific binding was defined by 10xe2x88x925 M diazepam and typically represented less than 5% of the total binding. Assays were incubated to equilibrium for 1 hour at 4xc2x0 C. and harvested onto GF/C uni-filters (Packard) by filtration using a Packard harvester and washing with ice-cold wash buffer (50 mM Tris; pH 7.5). After drying, filter-retained radioactivity was detected by liquid scintillation counting. Ki values were calculated using Excel-Fit (Microsoft) and are the means of two determinations.
The compounds of the accompanying examples were tested in the above-described assay, and all were found to possess a Ki value for displacement of [3H]Ro 15-1788 from xcex15 subunits of the rat GABA A receptor of 100 nM or less. In a preferred embodiment the compounds of the invention are binding selective for the xcex15 subunit relative to the xcex11, xcex12, and xcex11 subunit with an affinity of less then 15 nM.
The following specific data according to the test method described above for the especially preferred compounds of the present invention are presented in Table I below.
The compounds of formula I as well as their pharmaceutically acceptable acid addition salts can be used as medicaments, e.g. in the form of pharmaceutical compositions. The pharmaceutical compositions can be administered orally, e.g. in the form of tablets, coated tablets, dragxc3xa9es, hard and soft gelatin capsules, solutions, emulsions or suspensions. The administration can, however, also be effected rectally, e.g. in the form of suppositories, or parenterally, e.g. in the form of injection solutions.
The compounds of formula I and their pharmaceutically acceptable acid addition salts can be processed with pharmaceutically inert, inorganic or organic excipients for the production of tablets, coated tablets, dragxc3xa9es and hard gelatin capsules. Lactose, corn starch or derivatives thereof, talc, stearic acid or its salts etc. can be used as such excipients e.g. vegetables dragxc3xa9es and hard gelatin capsules. Suitable excipients for soft excipients e.g. for vegetable oils, waxes, fats, semisolid and liquid polyols etc.
Suitable excipients for the manufacture of solutions and syrups are e.g. water, polyols, saccharose, invert sugar, glucose etc.
Suitable excipients for injection solutions are e.g. water, alcohols, polyols, glycerol, vegetable oils etc.
Suitable excipients for suppositories are e.g. natural or hardened oils, waxes, fats, semi-liquid or liquid polyols etc.
Moreover, the pharmaceutical compositions can contain pharmaceutically acceptable preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorants, salts for varying the osmotic pressure, buffers, masking agents or antioxidants. They can also contain still other therapeutically valuable substances.
The dosage can vary within wide limits and will, of course, be fitted to the individual requirements in each particular case. In general, in the case of oral administration a daily dosage of about 10 to 1000 mg per person of a compound of general formula I should be appropriate, although the above upper limit can also be exceeded when necessary.
The following examples illustrate the present invention without limiting it. Unless stated to the contrary, all of the compounds listed as examples below were actually prepared and characterized as described. All temperatures are given in degrees Celsius.